Exposure to traffic-related air pollution such as diesel exhaust (DE) is associated with an increase in the level of reactive oxygen species (ROS) in multiple cell types. These ROS include the superoxide anion radical, hydrogen peroxide, lipid peroxides and hydroxyl radical. ROS can have deleterious effects on cells, but they are also known to be involved in normal transmembrane signaling and metabolic control in the cardiovascular system. For instance, two potent vasoconstrictors, angiotensin II and endothelin-1, can increase production of ROS by smooth muscle cell NADPH oxidase, thus causing an increase in the intracellular concentrations of superoxide and hydrogen peroxide. Superoxide can bind to nitric oxide (NO) to form peroxynitrite, which can cause cellular injury, but also limit the amount of NO available for vasorelaxation. ROS generated in this way can also oxidize tetrahydrobiopterin, a necessary cofactor for NO synthase (NOS), resulting in uncoupling of NOS and production of more superoxide and peroxynitrite. Consumption of NO by superoxide in this way can result in vasoconstriction. ROS are thus able to influence cardiovascular performance and vascular reactivity. Glutathione (GSH) is an abundant non-protein thiol which is a potent scavenger of ROS, including hydrogen peroxide, lipid peroxides, and importantly, peroxitrite (ONOO-). GSH is a tripeptide thiol present in millimolar concentrations in most cells. The first and rate-limiting step in GSH synthesis is carried out by the enzyme glutamate cysteine ligase (GCL), which is composed of two subunits, a catalytic subunit (GCLC) and a modifier or regulatory subunit (GCLM). Importantly, single nucleotide polymorphisms (SNPs) in both GCLC and GCLM have been shown to be important in myocardial infarction, as well as controlling vascular reactivity in humans. In this project, we will investigate the reasons for this effect of GCL by using a comparative approach. We will 1) use mouse models of differential GCL expression and activity to investigate its role in DE-induced changes in vascular reactivity 2), use cultured endothelial cells from these mice and from humans to investigate the underlying biochemical events responsible for GSH and DE-induced modulation of endothelial NO production; and 3) characterize genetically defined inbred strains of mice that have variability in their vascular responses to DE, and identify single nucleotide polymorphisms (SNPs) and quantitative trait loci that are associated with these changes in mice. Using these tools we will define the role of GSH synthesis in DE-induced changes in vascular reactivity, investigate the underlying pathophysiological mechanisms, and map and identify genetic determinants of DE-induced changes in vascular reactivity. These candidate genes will then be referred to Projects 1 and 2 where they will be further evaluated as potential genetic factors in DE-induced vascular abnormalities in humans.